The present invention pertains to vectors constructed in a prokaryotic cell for use in gene transfer to eukaryotic cells, vectors useful for making eukaryotic gene transfer vectors, and methods of making the same.
Gene transfer to eukaryotic cells has both in vivo and in vitro uses. As is well known, in vivo gene transfer to eukaryotic cells can be used to immunize a host, for therapeutic gene transfer to a host, and to study the biology of transferred genes in vivo. In vitro gene transfer to eukaryotic cells can be used to study simple and complex biological phenomena such as protein function, protein half-life, and gene-protein interactions. One preferred method for transferring genes to eukaryotic cells has been through the use of recombinant eukaryotic viruses. Although researchers and clinicians have enjoyed the many advantages of recombinant eukaryotic viruses for gene transfer to eukaryotic cells, the difficulty of constructing these viruses has impeded the rate at which new and useful gene transfer experiments and protocols have been developed.
Because of their large size, many recombinant eukaryotic viruses are produced via homologous recombination. Conventionally, homologous recombination used to generate large viral vectors has taken place in a host eukaryotic cell permissive for the growth of the recombinant virus (see, e.g., Berkner, BioTechniques, 6, 616-628 (1988)). Homologous recombination in eukaryotic cells, however, has at least two major drawbacks. The process is time consuming, and many preferred recombinant eukaryotic viral constructions are at a selective disadvantage relative to the predecessor eukaryotic viruses from which they are obtained. Therefore, if a skilled artisan attempts to create a new recombinant virus through the lengthy process of homologous recombination in a eukaryotic cell and fails to create the desired virus, that artisan is often unable to readily distinguish between the need to modify the construction technique and the possibility that the desired virus vector is not viable in the host cell. Accordingly, there is a need for new methods of generating eukaryotic gene transfer vectors.
Previous improvements in the generation of gene transfer vectors have included the use of yeast-based systems (Ketner et al., Proc. Nat""l. Acad. Sci. (USA), 91, 6186-6190 (1994)), plasmid-based systems (Chartier et al., J. Virology, 70, 4805-4810 (1996); Crouzet et al., WO 96/25506), and cosmid-based systems (Miyake et al., Proc. Nat""l. Acad. Sci. (USA), 93, 1320-1324 (1996)). While these systems can expedite the production of new recombinant eukaryotic viruses, additional flexibility and selection pressures are desired. The present invention provides a rapid and flexible method for producing new vectors, which can be used in gene transfer to eukaryotic cells in vitro and in vivo. The present invention also provides vectors modified for use in eukaryotic gene transfer, as well as methods and systems for using the same. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention provides a DNA vector comprising a portion of a eukaryotic viral genome comprising an ITR, a regulatable negative selection gene (NSG) or a stringently regulated growth discrimination gene (SRG), and a phage packaging site. The present inventive vector preferably comprises a full eukaryotic (e.g., adenoviral) amplicon. Moreover the regulatable negative selection gene or growth discrimination gene is preferably embedded within the portion of the eukaryotic viral genome of the present inventive vector such that a double recombination event with a second DNA vector removes the regulatable negative selection gene or growth discrimination gene at the same time that a DNA of interest is transferred into the present inventive vector. In another embodiment of the present inventive vector a positive selection gene is proximal to the NSG, which forms a dual selection cassette (DSC). The combination of negative and positive selection genes forms a dual selection cassette (DSC) that provides the skilled artisan with exquisite control of the homologous recombination system. The SRG can serve either a negative or positive selective function, or both. Accordingly, it is useful to have an SRG proximal to either a PSG or an NSG. SRGs that are adjacent or proximal to another selective gene are termed dual discrimination cassettes. Of course, the present inventive vector can also comprise other genetic elements, such as an independent positive selection gene that is not positionally associated with the NSG, DSC, or SRG and a bacterial origin of replication.
The present inventive vector optionally comprises additional advantageous elements. For example, the present inventive vector optionally comprises a deficient or conditionally deficient lambdoid origin of replication, which enhances the effectiveness of double homologous recombination. The present inventive vector is preferably configured such that the phage packaging site is proximal to an ITR of a eukaryotic viral amplicon, which allows for direct generation of an amplicon when the present inventive vector is encapsidated and transduced or infected into a suitable eukaryotic cell.
The present invention also provides for a library comprising or consisting of a multiplicity of the present inventive vector comprising a multiplicity of genetic elements that may be the same or different.
The present invention also provides a system for the generation of recombinant DNA vectors. Any embodiment of the present inventive vector can constitute a portion of the present inventive system. The present inventive system comprises at least a second DNA that comprises two DNA segments each of which have sufficient homology to the inventive vector to mediate homologous recombination and which flank or surround a DNA that is desirable to incorporate into the present inventive vector or into a portion of the eukaryotic viral DNA or amplicon that forms a portion of the present inventive vector. Certain embodiments of the present inventive system comprise a third DNA that can complement in trans a deficiency in a lambdoid origin of replication and, optionally, a fourth DNA that expresses a source of phage capsids that encapsidate intermediate or product vectors comprising the phage origin of replication. Either or both of the third and fourth DNAs of the inventive system can be optionally incorporated into the genome of a bacterial cell.
The present invention also provides a method of making and packaging a DNA vector. The inventive method comprises transfecting a bacterial cell with two DNA vectors that undergo homologous recombination to form a desired DNA vector. One vector comprises a phage packaging site, and an NSG, DSC, or SRG that is flanked by two DNA segments that mediate a double homologous recombination event with the second vector employed in the system. The double homologous recombination event places a DNA into the first vector and simultaneously removes the NSG, DSC, or SRG, and introduces a DNA from the second vector into the first vector. The method also includes the use of in vivo conditions that encapsidate the double homologously recombined product that contains a phage packaging site into a phage capsid. The present inventive method can also comprise infecting the encapsidated product vector into a population of cells under conditions such that the negative selection gene is active prior to harvesting the product vector from a lysate of the second cell, which serves to eliminate cells containing undesired DNA constructs from the population of cells from which the product vector is isolated.
The present inventive method optionally employs a first vector comprising a deficient or conditionally deficient lambdoid origin of replication. In this embodiment of the present invention, the deficiencies in the lambdoid origin of replication are complemented in trans during the period of time in which the homologous recombination event occurs, which enhances the effeciency of the process for several reasons.
The present invention also provides an improved method of gene transfer to eukaryotic cells. The improved method comprises using lambdid vectors (defined below) to generate novel recombinant vectors including recombinant eukaryotic viral vectors, that are capable of transferring genes to eukaryotic cells. Lambdid vectors, or portions thereof, also can be transduced into eukaryotic cells without the aid of (eukaryotic viral or phage) coat proteins. Lambdid vectors transduced into eukaryotic cells can generate new recombinant eukaryotic viral vectors, direct heterologous gene expression, or be used for other purposes. Additionally, lambdid vectors can be encapsidated into lambdoid capsids comprising chimeric lambdoid coat proteins capable of binding to eukaryotic cells. These lambdid vectors encapsidated into recombinant capsids can be used to directly transduce eukaryotic cells.
These and other features of the present invention are more fully described below.